CN109917128A - The method of anti-Procalcitonin antibody coating particle - Google Patents
The method of anti-Procalcitonin antibody coating particle Download PDFInfo
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- CN109917128A CN109917128A CN201910272441.XA CN201910272441A CN109917128A CN 109917128 A CN109917128 A CN 109917128A CN 201910272441 A CN201910272441 A CN 201910272441A CN 109917128 A CN109917128 A CN 109917128A
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Abstract
The present invention relates to antibody modification technical fields; specifically; it is related to a kind of method of anti-Procalcitonin antibody coating particle, this method comprises: the particle modification has tosyl, the antibody reacts to form covalent bond completion coating with the tosyl by the amino of itself.The present invention can be used direct one-step method coating that anti-PCT antibody is coated on magnetic bead, and method is simple, is coated with good process repeatability, high sensitivity.
Description
Technical field
The present invention relates to antibody modification technical fields, in particular to a kind of anti-Procalcitonin antibody coating particle
Method.
Background technique
Procalcitonin (procalcitonin, PCT) is the bacterium just found nineteen nineties, fungal infection
Specific marker, be a kind of calcitonin propetide of no hormonal activity, be made of 116 amino acid, molecular weight is about
13KD.It can gradually be cracked into the amino Procalcitonin (N-PCT) of 57 amino acid, 32 amino acid under the action of enzyme
The katacalein of calcitonin (calcitonin, CT) and 21 amino acid.Procalcitonin is calcitonin I gene on No. 11 chromosomes
(CALC-I) expression product.There is no infection, the outer CALC-I expression of thyroid gland is suppressed, and is mainly limited to
The a degree of expression of the neuroendocrine cell of thyroid gland and lung；The various tissue various kinds of cell type of bacterium infection inducing systemic
CALC-I expression and Procalcitonin continuity release.Recent study show serious systemic bacterium, fungi, helminth,
In terms of acute malaria infection, the diagnosis of systemic inflammatory response syndrome (SIRS), Multiple Organ Failure Syndrome (MODS),
Procalcitonin is a New Set with highly sensitive specificity.
Procalcitonin has 116 amino acid altogether, can be divided into three sections according to structural domain and function.1-57 are its N sections.
N-terminal can start to be sheared at 57 in vivo, generate the 60-116 amino acids containing calcitonin.Meanwhile it also can be by 91
Proteolytic cleavage, forms calcitonin and anticalcium element is former (katacalcin of 96-116 amino acids).Therefore it is deposited in human serum
Segment after Procalcitonin is whole and its a variety of shearing, and these segments played in human body it is different with Procalcitonin
The physiological function of sample.
The detection common laboratory method of PCT at present has radioimmunology analytic approach (RIA), chemiluminescence immunoassay
(CLIA), colloidal gold colorimetric method (GICA) and enzyme-linked immunization (ELISA) etc..It is inspection that PCT, which is fixed in solid phase, such as magnetic bead,
Survey its conventional technical means.Its method for coating is usually to pass through first to be coated with the antibody that can identify PCT antibody, or packet
It can be identified the antibody of PCT mark molecule by one, or after activating certain groups by EDC etc., then be coated with PCT antibody, this
The a series of problems such as a little methods are cumbersome, and process is not easy to control, and final caused difference between batch is larger.
In view of this, the present invention is specifically proposed.
Summary of the invention
The technical problems to be solved by the present invention are: making up above-mentioned the deficiencies in the prior art, a kind of anti-Procalcitonin is proposed
The method that antibody is coated with particle, the method for coating have signal-to-noise ratio and luminous value high, and coating process is simpler, does not need complicated instrument
The advantages that device.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The present invention relates to a kind of methods of anti-Procalcitonin antibody coating particle, comprising:
The particle modification has tosyl, and the antibody reacts shape with the tosyl by the amino of itself
It completes to be coated at covalent bond.
This method selection for comparing other groups, may make coated anti-by the antibody of the fixed Procalcitonin of amino
Body has better signal-to-noise ratio and luminous value.
According to an aspect of the present invention, the particle that method as above is coated with is further related to.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is experimental principle schematic diagram of the invention.
Specific embodiment
The present invention relates to a kind of methods of anti-Procalcitonin antibody coating particle, comprising:
The particle modification has tosyl, and the antibody reacts shape with the tosyl by the amino of itself
It completes to be coated at covalent bond.
The group that the magnetic bead surfaces that the present invention uses have is the p-toluenesulfonyl ,-NH of the group and antibody2Group exists
It under conditions of certain, is chemically reacted, forms covalent bond, Procalcitonin (procalcitonin, PCT) antibody coupling is existed
Magnetic bead surfaces.Its specific coupling mechanism is as shown in Figure 1.
As it is used herein, " particle " refer to can be discrete wisp of various shapes, for example sphere (such as
Pearl), capsule, polyhedron etc..Particle can be macroscopic view or microcosmic, such as particle or nano particle.
" particle " used herein, especially " enrichment pearl " can be manufactured by any amount of known materials.This material
Example include: inorganic matter, natural polymer and synthetic polymer.The specific example of these materials includes: cellulose, cellulose
Derivative, acrylic resin, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, vinyl and acrylamide are total to
Polymers, polystyrene, polyacrylamide, latex gel, glucan, rubber, silica gel, plastics, nitrocellulose, natural sponge,
Silica gel, control wells glass (control pore glass), metal, cross-link dextran (such as Sephadex TM), agarose are solidifying
Glue (Sepharose TM) and other solid supports well known by persons skilled in the art.
Particle can be non magnetic or magnetic.Magnetic-particle may include ferromagnetic material, and ferromagnetic material can
To be Fe, Ni, Co, iron oxide etc..
Other groups are not used between the tosyl and the amino in some embodiments to be attached；
I.e. the present invention can be coupled by one-step method.
In some embodiments, the particle is γ Fe2O3Or Fe3O4Magnetic nano-particle or they and organic polymer
The complex of material.
High-molecular organic material can be the chelate containing organic anion added in the synthesis process, such as lemon
Acid, glucose, dimercaptosuccinic acid, Phosphorylcholine etc..
In some embodiments, if not illustrating, the magnetic bead particles partial size is 1.5um, surface tosyl
Density be 150nmol/mg, the mass concentration of magnetic bead aqueous solution is 100mg/ml；Antibody is aqueous solution, and mass concentration is not
Less than 2mg/mL.
During coating, the mass ratio of magnetic bead and the antibody is 25:1~100:1；Also can choose 30:1,35:1,
40:1,45:1,50:1,55:1,60:1,65:1,70:1,75:1,80:1,85:1,90:1 or 95:1.
The quality of particle is based on particle dry weight, in some embodiments, the coating buffering used in the process of the coating
Contain ammonium sulfate, cationic detergent, and pH=7.00~9.00 of the coating buffer in liquid；PH also can choose 7.5,
8,8.5 etc..
In some embodiments, concentration of the ammonium sulfate in the coating buffer is 0.5M~2M；It can be with
Select 1.0M or 1.5M.
In some embodiments, the cationic detergent is illustratively selected from following substance:
Containing alkyl quaternary ammonium compound, such as: trimethyl cetyl ammonium bromide (CTAB), trimethyldodecane base ammonium bromide；
Containing heteroatomic quaternary ammonium salt, such as: the quaternary ammonium salt of oxygen-containing, nitrogen, element sulphur；
Quaternary ammonium salt containing phenyl ring, such as: clean youngster is gone out, bromogeramine；
Quaternary ammonium salt containing heterocycle, such as: morpholine ring, pyridine ring, imidazole ring, piperazine ring, quinoline ring；
The quaternary ammonium salt of ammonium salt type, such as: Priminox series.
In some embodiments, the cationic detergent is CTAB.
In some embodiments, concentration of the CTAB in the coating buffer is 0.7g/L~1.3g/L；Also
It can choose 0.9g/L, 1g/L, 1.1g/L.
The one side effect of the cationic detergent and ammonium sulfate of suitable concentration is appropriate denatured antibody, exposes its ammonia
Base, and then obtain preferably coating effect.
Term " buffer " as used herein refers to aqueous solution or composition, when acid or alkali are added in the solution or composition
When, the aqueous solution or composition resist the variation in pH.This resistance to pH variation is the resiliency due to such solution
Matter.Therefore, show that the solution of buffers active or composition are referred to as buffer or buffer solution.Buffer does not have nothing generally
The ability of the pH for maintaining solution or composition of limit.On the contrary, they are generally possible to maintain the pH in particular range, such as pH
7–pH 9.Generally, buffer be able to maintain that in the upper and lower logarithm of its pKa pH (see, for example, Mohan,
Buffers, A guide for the preparation and use of buffers in biological systems,
CALBIOCHEM, 1999).Buffer and buffer solution are generally prepared by buffer salt or nonionic buffer composition.
In this application, the buffer components preferably used in buffer are coated with as the carbonate of 40mM~60mM, concentration
It can choose 45mM, 50mM, 55mM.
The substantially non-confrontational body coating of carbonate has an adverse effect.
According to an aspect of the present invention, the particle that method as above is coated with is further related to.
The present invention is by groping the quality pack of magnetic bead and antibody by ammonium sulfate concentrations in ratio, coating buffer, pH value, no
Anti- PCT antibody is coated on magnetic bead by the conditions such as the detergent of same type, direct one-step method coating, and method is simple, coating technique
It is reproducible, high sensitivity.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment 1
Surface coupling there is into the ratio of the magnetic bead and PCT antibody of tosyl with mass ratio for 50:1, wherein toluene sulphur
Acyl group and antibody mass ratio are 7500nmol:1mg, are containing 1M ammonium sulfate, the 50mM carbonic acid that 0.1w/v%CTAB, pH are 9.0
It is coated in salt buffer.
Embodiment 2
Surface coupling there is into the ratio of the magnetic bead and PCT antibody of tosyl with mass ratio for 25:1, is containing 0.5M
It is coated in ammonium sulfate, the 60mM carbonate buffer solution that 0.07w/v%CTAB, pH are 8.0.
Embodiment 3
Surface coupling there is into the ratio of the magnetic bead and PCT antibody of tosyl with mass ratio for 100:1, is containing 2M sulphur
It is coated in sour ammonium, the 40mM carbonate buffer solution that 0.13w/v%CTAB, pH are 7.0.
Comparative example
Comparative example 1:
Selection magnetic bead particles diameter is 3um, and the density of surface tosyl is 80nmol/mg, the quality of magnetic bead aqueous solution
Concentration is the tosyl magnetic bead of 100mg/ml, for being mutually coupled with PCT antibody.
In addition to this condition is same as Example 1, the mole including tosyl and antibody mass ratio.
Comparative example 2:
Surface coupling is selected to have the magnetic bead of-COOH (carboxyl) group, feature is that partial size is 1.5um, and the carboxyl on surface is close
Degree is 25nmol/mg, mass concentration 100mg/mL, for being mutually coupled with PCT antibody amino groups.
In addition to this condition is same as Example 1, the mole including carboxyl and antibody mass ratio.
Experimental example 1
1. being coated with three batches of magnetic beads according to embodiment 1 and being not optimised method (the 50mM carbonate buffer solution of pH 9.0) comparison.
Detection method is as follows: magnetic bead takes 24ug magnetic bead after being coated with, and 30ul is added and contains various concentration Procalcitonin
Sample adds the anti-Procalcitonin antibody for the label alkaline phosphatase that 50ul concentration is 0.25ug/ml, after mixing, 37 DEG C of knots
It closes 10 minutes, substrate solution is added in washing, after incubation, detects luminous value.Calculate the luminous value and dilution sample for there are concentration samples
Luminous value ratio, i.e. signal-to-noise ratio, noise compare so as to see who is superior as the foundation for comparing coating condition.
Conclusion:
The coefficient of variation (CV) mean value is down to 1.22% by 8.64% after coating system optimization, reduces by 7 times, wraps after illustrating optimization
The difference between batch of quilt is stablized.
After being coated with system optimization, background is reduced to 687 by 2701, reduces 3.9 times, after illustrating optimization system, coated
Sensitivity has been improved.
2. the optimization of the condition of coating:
It is coated with according to the method for coating of embodiment 1 using magnetic bead and antibody different quality ratio, discovery mass ratio is
When 50:1, signal-to-noise ratio highest.Optimal packet is by quality than range in 25:1-100:1.
3. being coated with using the ammonium sulfate of different molar concentrations according to the method for coating of embodiment 1, finding ammonium sulfate
Optium concentration is 0.5M-2M.
4., using different pH value coating buffers, finding Optimal pH between 7.00-9.00 according to the method for coating of embodiment 1.
Conclusion: it can be seen from the results above that coating efficiency is very strong to the dependence of pH value, find Optimal pH in 7.00-
Between 9.00.
5. different types of cationic detergent is added in coating buffer according to the method for coating of embodiment 1, to coated shadow
It rings it is obvious that filtering out the cationic detergent of optimal type.
Conclusion: in the cationic detergent of 4 seed types, coating background is can be effectively reduced in cationic detergent, drops background
Efficiency is up to 3.7 times.
Experimental example 2
Surface coupling there is into the ratio of the magnetic bead and PCT antibody of tosyl with mass ratio for 25:1, is containing 0.5M
It is coated in ammonium sulfate, the 60mM carbonate buffer solution that 0.07w/v%CTAB, pH are 8.0.
Experimental example 3
Surface coupling there is into the ratio of the magnetic bead and PCT antibody of tosyl with mass ratio for 100:1, is containing 2M sulphur
It is coated in sour ammonium, the 40mM carbonate buffer solution that 0.13w/v%CTAB, pH are 7.0.
Comparative example experiment 1
Selection magnetic bead particles diameter is 3um, and the density of surface tosyl is 80nmol/mg, the quality of magnetic bead aqueous solution
Concentration is the tosyl magnetic bead of 100mg/ml, for being mutually coupled with PCT antibody.
In addition to this condition is same as Example 1, the mole including tosyl and antibody mass ratio.
Comparative example experiment 2
Surface coupling is selected to have the magnetic bead of-COOH (carboxyl) group, feature is that partial size is 1.5um, and the carboxyl on surface is close
Degree is 25nmol/mg, mass concentration 100mg/mL, for being mutually coupled with PCT antibody amino groups.
In addition to this condition is same as Example 1, the mole including carboxyl and antibody mass ratio.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Claims (10)
1. the method for anti-Procalcitonin antibody coating particle characterized by comprising
The particle modification has tosyl, and the antibody reacts to be formed altogether with the tosyl by the amino of itself
Valence link completes coating.
2. the method according to claim 1, wherein the particle is γ Fe2O3Or Fe3O4Magnetic nano-particle,
Or the complex of they and high-molecular organic material.
3. the method according to claim 1, wherein the mass ratio of the particle and the antibody be 25:1~
100:1；It is preferred that 50:1.
4. the method according to claim 1, wherein containing in coating buffer used in the process of the coating
Ammonium sulfate, cationic detergent, and the pH=7.00-9.00 of the coating buffer.
5. according to the method described in claim 4, it is characterized in that, concentration of the ammonium sulfate in the coating buffer is
0.5M~2M.
6. according to the method described in claim 4, it is characterized in that, the cationic detergent is selected from chemical combination containing quaternary ammonium alkyl
Object, the quaternary ammonium salt containing heteroatomic quaternary ammonium salt, the quaternary ammonium salt containing phenyl ring, the quaternary ammonium salt containing heterocycle and ammonium salt type.
7. according to the method described in claim 6, it is characterized in that, the cationic detergent is CTAB.
8. the method according to the description of claim 7 is characterized in that concentration of the CTAB in the coating buffer is
0.7g/L~1.3g/L.
9. according to the method described in claim 4, it is characterized in that, the buffer components used in the coating buffer is 40mM
The carbonate of~60mM.
10. the particle that any one of claim 1~9 the method is coated with.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110596373A (en) * | 2019-09-19 | 2019-12-20 | 潍坊市康华生物技术有限公司 | Method for coupling magnetic particles and antibody |
| CN111579772A (en) * | 2020-05-28 | 2020-08-25 | 珠海丽珠试剂股份有限公司 | Detection reagent based on immunomagnetic bead method, preparation method thereof, kit and detection method |
| CN112710857A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | f-beta-hCG protein detection kit and detection method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110596373A (en) * | 2019-09-19 | 2019-12-20 | 潍坊市康华生物技术有限公司 | Method for coupling magnetic particles and antibody |
| CN111579772A (en) * | 2020-05-28 | 2020-08-25 | 珠海丽珠试剂股份有限公司 | Detection reagent based on immunomagnetic bead method, preparation method thereof, kit and detection method |
| CN111579772B (en) * | 2020-05-28 | 2022-04-08 | 珠海丽珠试剂股份有限公司 | Detection reagent based on immunomagnetic bead method, preparation method thereof, kit and detection method |
| CN112710857A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | f-beta-hCG protein detection kit and detection method |
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